. The PI proposes to develop extremely sensitive assays for the simultaneous detection of pathogenic retroviruses in blood and donated tissue. These assays will utilize binary probes that hybridize to adjacent positions on retroviral target RNA. The probes are then ligated to each other, in a target-dependent manner, to form a replicatable reporter RNA. This RNA is then amplified as much as 100 billion-fold in a brief incubation with Qb replicase (bacteriophage), signaling the presence of the target. A single HIV-1 infected cell can be detected in a sample containing 100,000 healthy cells. Binary probes that are specific for HIV-1, HIV-2, HTLV-I, and HTLV-II will be combined into a multiplex assay. The amplified reporter RNAs will be identified with the aid of molecular beacons, which are nonfluorescent detector molecules that undergo a fluorogenic conformational change in the presence of a particular RNA. Four different molecular beacons, each fluorescing at a different wavelength, and each specific for a different reporter RNA, will be included in the amplification reaction. The presence of a particular retroviral species will be indicated by the color of the fluorescence. Binary probes and molecular beacons will also be adapted for use in the detection of drug-resistance mutations that become prominent in a patient's retroviral population in response to the use of antiviral drugs. Two assay designs will be explored: one utilizes the ability of a ligase to discriminate against the presence of a mismatched nucleotide at the binary probe ligation junction; and the other utilizes the ability of molecular beacons to reject targets that differ by a single nucleotide. These assays will identify drug-resistant mutants early in the course of treatment, enabling the physician to alter the therapy.